Production and function of E coli vesicles

描述（由申请人提供）：大肠杆菌，以及迄今为止研究的所有革兰氏阴性亲缘菌，经历外膜的囊泡或挤压过程。囊泡已经被证明能够与细菌和真核生物的膜融合，在这个过程中传递可溶性和膜成分。虽然它们的存在已经被认识了几十年，但细菌外膜囊泡尚未在基本的遗传和生化水平上进行研究。在初步工作中，已经开发了从大肠杆菌中分离和纯化囊泡的方法。第一个目标是用遗传方法来阐明产生囊泡的细胞机制。随机产生的大肠杆菌突变体将被筛选在囊泡产生方面的缺陷。第二个目标是在正常生理条件和应激生长条件下，囊泡如何有益于“母细胞”。囊泡在细胞间交流的能力可能有助于在竞争激烈的生长环境中传播遗传信息。利用生化分析，我们将研究蛋白质、脂质和核酸的囊泡介导传播。人工脂质体在体外已被证明与细菌膜融合，这些实验为开始研究囊泡膜特性提供了基础，这些特性可能对融合很重要。在紧张的生长条件下，囊泡途径可能被选择以允许外膜的快速重塑。在冷休克后，囊泡对脂多糖组成的剧烈变化的贡献将被分析。此外，囊泡突变体将用于研究细菌对不同环境胁迫的反应过程中囊泡的功能。提案的目标是不同的，不依赖于彼此，但建立在彼此的基础上，以解决项目假设。这种遗传学和生物化学的强大结合将为细菌生理学这一基础领域的未来发现奠定基础。预计这些研究将揭示膜动力学的一般原理，同时也突出了细菌膜独特的新概念。

DESCRIPTION (provided by applicant): Escherichia coli, as well as all of its gram-negative relatives studied to date, undergo a process of vesiculation, or pinching-off, of the outer membrane. Vesicles have been shown to be capable of fusion with both bacterial and eukaryotic membranes, delivering soluble and membrane components during this process. Although their presence has been recognized for decades, bacterial outer membrane vesicles have yet to be investigated at a basic genetic and biochemical level. In preliminary work, methods have been developed to isolate and purify vesicles from E. coli. The first objective is a genetic approach to elucidate the cellular machinery that produces vesicles. Randomly generated E. coli mutants will be screened for defects in vesicle production. The second objective is to how vesicles benefit the "mother cell" under normal physiological conditions and under stressful growth conditions. The ability of vesicles to communicate between cells may help in a competitive growth environment and to disseminate genetic information. Using biochemical assays, we will investigate vesicle-mediated transmission of proteins, lipids and nucleic acids. Artificial liposomes have been shown to fuse with bacterial membranes in vitro and these experiments provide a basis to begin studying vesicle membrane characteristics that may be important for fusion. Under stressful growth conditions, the vesicle pathway may be co-opted to allow quick remodeling of the outer membrane. The contribution of vesicles to the dramatic switch in lipopolysaccharide composition after cold shock will be analyzed. Further, vesiculation mutants will be used to investigate the function of vesicles during a bacterial response to diverse environmental stresses. The aims of the proposal are distinct and do not depend on one another, yet build on each other to address the project hypotheses. This powerful combination of genetics and biochemistry will set a foundation for future discoveries in this basic area of bacterial physiology. It is anticipated that these studies will reveal general principles of membrane dynamics, while also highlighting new concepts unique to bacterial membranes.